We have previously shown that Ntr1 and Ntr2 form a stable complex, which further interacts with Prp43 to form the NTR complex through the interaction of Ntr1 and Prp43. The NTR complex is functional in mediating the disassembly of the lariat intron-containing spliceosome (37
). We demonstrate here that the interaction between Prp43 and the Ntr1-Ntr2 complex is dynamic, and Prp43 could readily dissociate from Ntr1-Ntr2 upon dilution of the complex. This provides an explanation for the presence of a fraction of Ntr1-Ntr2 complex in the splicing extract, considering Prp43 is in great excess.
In the absence of Prp43, the Ntr1-Ntr2 complex could also bind to the spliceosome and recruit Prp43 to the spliceosome upon its addition. Neither the binding of Ntr1-Ntr2 nor the binding of Prp43 to the spliceosome requires ATP. ATP is required only for the disassembly reaction. Specifically, hydrolysis of ATP by Prp43 is required, because the D215A ATPase mutant of Prp43 is not functional in catalyzing disassembly of the Ntr1-associated spliceosome. Similar scenarios have been shown for Prp2 and Prp16, for which ATP is required only for their function in mediating conformational rearrangements of the spliceosome but not for their binding to the spliceosome (13
). Prp2 has been shown to interact with the G-patch domain-containing protein Spp2 to promote the step one reaction (26
). Spp2 is required for the function of Prp2 and presumably plays a role in the recruitment of Prp2 to the spliceosome similar to the function of Ntr1-Ntr2 in recruiting Prp43.
In addition to dynamic interactions with Prp43, we have shown here that the Ntr1-Ntr2 complex also interacts with U5 snRNP in a dynamic manner. A small fraction of U5 coprecipitated with Ntr1, Ntr2, and Prp43 from splicing extracts. Mixing of U5-depleted extracts and NTR-depleted extracts resulted in reassociation of U5 and NTR. Coprecipitation of U5 with Prp43 has previously been demonstrated (10
). We show that Prp43 does not directly interact with U5 and is associated with U5 through its association with Ntr1-Ntr2. It has also been reported recently that both Ntr1 and Ntr2 coprecipitated small amounts of U2, U5, and U6 by using TAP-tagged Ntr1 and Ntr2 extracts (2
). Nevertheless, the fraction of U5 coprecipitated from the extract was greater than that of U2 or U6. Coprecipitation of amounts of U2 and U6 in the reported experiment larger than those in ours might be due to higher levels of the endogenous spliceosome in their extracts.
Proteomic analyses have revealed that protein complexes are widely present in the cell. However, the stoichiometry of the components in individual complex could not be assessed. A recent analysis of Yju2 protein has revealed its function in promoting the first catalytic step of splicing, despite its association with NTC, which is involved in spliceosome activation (19
). Yju2 also interacts with NTC in a dynamic manner and may be recruited to the spliceosome by NTC through such dynamic interactions. In view of this, dynamic interactions might represent a significant portion of interactions in the proteomic database. Furthermore, dynamic interactions between protein components might be a general property for the recruitment of components to macromolecular complexes in various cellular pathways.
Several lines of evidence suggest that the association of Ntr1-Ntr2 with U5 is mediated through the interaction of Ntr2 with U5 snRNP. First, polyclonal antibodies against Ntr2 blocked the association of U5 with NTR complex as well as binding of NTR to the spliceosome. Second, genetic depletion of Ntr2 uncoupled the association of Ntr1 with U5 and with the spliceosome. Third, Ntr2 could bind to the spliceosome and also associate with U5 independently of Ntr1. Finally, Ntr2 interacts with U5 component Brr2 in two-hybrid assays. Thus, Ntr2 is essential and sufficient for the interaction of NTR with U5 and binding of NTR to the spliceosome, and such interactions are likely mediated through its interaction with Brr2. A diagram illustrating how dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 might mediate spliceosome disassembly is shown in Fig. .
A diagram illustrating the interactions of Ntr1-Ntr2 with Prp43 and with U5 to mediate spliceosome disassembly. Double arrows indicate equilibrium between association and dissociation forms.
Association of NTR with U5 but not with U4/U6.U5 tri-snRNP indicates that the interaction of U4/U6 prevents U5 from interacting with NTR. Protein-protein interactions within the human tri-snRNP have been analyzed in detail and demonstrate a key role of human Snu66 (hSnu66) and hPrp6 in bridging U4/U6 and U5 in the formation of tri-snRNP (17
). hPrp6 interacts with tri-snRNP-specific hSnu66 and U4/U6-specific hPrp3 and hPrp31 besides interacting with other U5 proteins and is required for tri-snRNP stability. Although hSnu66 also interacts with both U4/U6 and U5 proteins, it is not required for tri-snRNP stability (21
). In view of this interaction network, Prp6 might play a role in modulating the structure of U5 snRNP for interactions with U4/U6 and with NTR.
was also identified as SPP382
in a genetic screen for suppressors of prp38
), which causes a temperature-sensitive growth defect and, in vitro, results in slow release of U1 and U4 during spliceosome activation (40
). Mutant alleles of SPP382
show suppression of both prp38
, suggesting a link between NTR1
and U5. A mutant of AAR2
that encodes a U5 protein also acts as a prp38
suppressor, further supporting the U5-NTR linkage. Aar2 is specifically associated with U5 but not with the U4/U6.U5 tri-snRNP and has been shown to function as a recycling factor, possibly required for regeneration of the tri-snRNP during spliceosome cycling (11
). Interestingly, several mutations in PRP43
affecting the ATPase activity also suppress the prp38
growth defect (24
). These results suggest that reducing the rate of spliceosome cycling might partially compensate for impaired spliceosome assembly. The DEAH-box ATPases Prp16 and Prp22 have been demonstrated to play roles in modulating splicing fidelity by coupling ATP hydrolysis with a discard pathway (5
), but whether they are directly involved in disposing of the defective spliceosome is not known. Since NTR is recruited to the spliceosome by U5, it is conceivable that NTR can also bind to the spliceosome at earlier steps if spliceosome assembly is retarded. It will be of interest to see whether Prp43 can function to scavenge the impaired spliceosome in the discard pathway.
Proteomic analysis of mammalian splicing complexes has revealed the presence of a 35S, U5-containing particle in nuclear extracts. The complex also contains components of the Prp19-associated complex and other uncharacterized factors, many of which are present in the mature 45S spliceosome. It has been proposed that the 35S complex represents a disassembly intermediate of the spliceosome (20
). Unlike in mammalian extracts, the U5-NTC complex has not been detected in yeast. Immunoprecipitation of NTC coprecipitated minute amounts of U2, U5, and U6 (Fig. , lane 4), presumably representing the endogenous spliceosome, but has never coprecipitated a significantly greater amount of U5. In contrast, U5 was found to associate with NTR almost quantitatively in spliceosome disassembly assays (Fig. ). Although this suggests that U5 might be dissociated from the spliceosome in association with NTR, we cannot exclude the possibility that the association occurred after the disassembly due to dynamic interactions of U5 and NTR. Nevertheless, mammalian and yeast spliceosomes might undergo disassembly via distinct mechanisms.